The hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells secretory functions Factors in hypothalamus increase the rate of ACTH secretion by the pituitary gland A physiologic corticotropin releasing factor (CRF), i.e., ovine CRF (oCRF), was characterized in 1981 and disclosed in U.S. Pat. No. 4,415,558 to have the formula: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His -Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln -Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH.sub.2.
Rat CRF(rCRF) has been characterized as having the formula: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr -Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu -Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH.sub.2 and may alternatively be referred to as rat Amunine. The formula of human CRF has been determined to be the same as that of rCRF. Synthetic rCRF and oCRF stimulate ACTH and .beta.-endorphin activities in vitro and in vivo and substantially lower blood pressure for an extended time period Both of these hypophysiotropic factors have been reproduced by total synthesis, and analogs of the native structures have been synthesized and tested. Since these discoveries, CRF peptide analogs have been synthesized which have a high binding affinity for CRF-receptors and which are more potent and longer acting than the native hormones; research in this area is continuing.
Physiologists have known the cytotoxic effects of ricin and gelonin since 1974 and 1980, respectively. Both ricin A chain, the chain that carries the cytotoxic activity, and gelonin are inhibitors of protein synthesis and have similar molecular weights and functional properties. Ricin A chain is the more potent of the two. Both cytotoxins previously have been conjugated to antibodies and are described in the literature. Gelonin is described in J. Biol. Chem., 255, 14, 6947-6953 (1980).
Cytotoxins are generally only toxic to a cell when present in the cytoplasm. It is known to deliver cytotoxins to the cytoplasm by contacting an intact cell with a cytotoxic conjugate comprising a cytotoxin and a bioactive molecule (e.g. immunoglobulin or hormone). Such cytotoxic conjugates have an affinity for a target cell which expresses on its surface a molecule which forms a binding pair with the bioactive molecule of the conjugate (e.g., an antigen or hormone receptor) and the conjugate is transported to the cytoplasm, presumably by endocytosis See Uhr et al., U.S. Pat. No. 4,664,911 and Bacha et al., U.S. Pat. No. 4,468,382.
Importantly, the bioactive component of the cytotoxic conjugate must be linked to the cytotoxin without adversely affecting binding affinity of the former. This problem is of more concern with respect to hormones, which characteristically are small peptides having a molecular weight of less than 5,000-10,000 daltons, whereas immunoglobulins are relative large proteins having molecular weights typically greater than 100,000 daltons. Consequently, the probability of successfully conjugating a bioactive molecule to a cytotoxin without adversely affecting the binding capacity of the bioactive molecule is greater in the case of immunoglobulin-cytotoxic conjugates than in the case of hormone-cytotoxic conjugates. In other words, as compared to a peptide hormone, the larger immunoglobulin protein will, on the average, have more amino acid residues available for linkage at positions which will not adversely affect its bioactivity (i.e., its capacity for binding antigen with high affinity). Thus, with respect to making hormone-cytotoxin conjugates having high binding affinity for the native receptor, if conjugation occurs through an amino-acid residue near the portion of the hormone which is critical for high affinity binding, the resultant conjugate will lack substantial capacity for binding native hormone receptor.
Furthermore, where more than one amino acid residue of the hormone is capable of forming a covalent linkage with a linker employed to form a hormone-cytotoxin conjugate, undesirable cross-linked polymers of the hormone may be formed, which may substantially preclude the capacity to bind the native hormone receptor.
Heterobifunctional linkers have been used to cause a linker to bind to particular amino acid residues (e.g., histidines) of one member of the conjugate. Bacha et al., supra, disclose a disulfide-linked, thyrotropin releasing hormone-cytotoxin conjugate which is prepared by: (i) disulfide-derivatizing the hormone (through available histidine residues) using a heterobifunctional coupling agent, (ii) disulfide-derivatizing a cytotoxin using a different heterobifunctional coupling agent, and (iii) carrying out a disulfide replacement reaction between the derivatized hormone (provided as a sulfhydryl derivative) and the derivatized cytotoxin (provided as a disulfide derivative) to form the hormone-cytotoxin conjugate.
But, despite the use of such heterobifunctional linkers it can not be predicted that the resultant hormone-cytotoxin conjugates will retain high-affinity binding capacity for the hormone's native receptor, since conjugation is directed to e.g., all available histidine residues in the peptide hormone. Therefore, if a portion of the hormone necessary for high-affinity binding of receptor comprises one or more histidine residues, the resulting conjugate will lack such binding capacity due to the addition bulk or steric hindrance presented by a massive cytotoxin molecule.
Moreover, where the hormone has more than one amino acid residue capable of reacting with a coupling agent employed in the conjugation, and thus is caused to be coupled at more than one position, upon conjugation of the "derivatized" hormone to a cytotoxin, the hormone may be linked in a sterically hindered conformation due to conjugation at the more than one "derivatized" residues of the hormone, or may be linked to more than one cytotoxin molecule, again, undesirably placing conformational constraints on the hormone which may adversely affect binding. Likewise, if a hormone has more than one reactive residue, it is probable that undesirable polymers of hormone-toxin conjugate will result (e.g., hormone-toxin-hormone.sub.n), which polymers analogously lack high affinity binding capacity.